Vmax Regulation through Domain and Subunit Changes in the Active Form of Phosphoglycerate Dehydrogen

1
Vmax Regulation through Domain and Subunit
Changes in the Active Form of Phosphoglycerate
Dehydrogenase
James R. Thompson, Jessica K. Bell, Judy Bratt,
Gregory A. Grant, and Leonard J. Banaszak
Presented by Thai Vo Spring 2006
2
(No Transcript)
3
(No Transcript)
4
Phospholipid and Tryptophan Metabolism pathway

• Tryptophan is an important amino acid in
  increasing the quality of grain

5
D-2-hydroxyacid dehydrogenase family
Tetramer
Dimers
D-lactate dehydrogenase
3-Phosphoglycerate Dehydrogenase
D-glycerate dehydrogenase
Trimer
Formate Dehydrogenase
6
(No Transcript)
7
Purification

• E. coli was grown in 2x YT broth ampicillin
• Induced with 1.5 mM isopropyl thigalatoside
• Lyse the cells by sonication
• Centrifugation
• Bring up saturation up to 70
• Column Chromatography
• Submersion in Seleniomethionine solution
• Test for activity
• Electrospray mass spectra

8
Crystallization

• Hanging drop vapor diffusion
• Analyzed via MAD phasing technique

9
Multiwavelength Anomalous Diffraction Phasing
10
Structural comparision between active and Inh-PGDH
11
RBD domain
BlackPGDH
Gray Inh-PGDH
12
Subunit A
Blue PGDH
RedInh-PGDH
13
Interface between NBD and SBD-RBD
14
Active Site of PGDH
15
Conclusion

• Vmax regulation of PGDH involve tertiary
  conformational changes and domain-domain and
  subunit reorientation.
• RBD/SBD behave as a single unit, conformational
  differences occurs at SB-NBD hinge
• Changes in the clef opening is needed for
  catalysis
• Serine cause negative cooperative process for
  ligand
• Process can represent a general mechanism among
  an entire group enzymes containing ACT domain

16
Thanks to

• Dr. Bell
• Wazo Myint
• Tony Downs
• Jacob Neals